1. Micropropagation is a process of rapidly multiplying plant materials using modern tissue culture methods. It involves 5 main stages - selection of stock plants, initiation and establishment of culture, multiplication of shoots, rooting of shoots, and establishment of plantlets. 2. There are 4 main approaches to micropropagation - multiplication by axillary buds/apical shoots, multiplication by adventitious shoots, organogenesis, and somatic embryogenesis. Each approach utilizes specific plant tissues and culture conditions. 3. Micropropagation has many applications including high rate propagation, production of disease-free plants, and generation of genetically modified plants. However, it also has disadvantages such as contamination, genetic variability, and vitrification of shoots.

1. Shehzadi Fatima
University Of
Sargodha

2. Micropropagation
 Plants can be propagated by sexual (through generation of
seeds) or asexual (through multiplication of vegetative
parts) means.
 Clonal propagation refers to the process of asexual
reproduction by multiplication of genetically identical
copies of individual plants, where the term clone is used to
represent a plant population derived from a single
individual by asexual reproduction.
 In vitro clonal propagation through tissue culture is
referred to as micro propagation.
Micropropagation is the practice of rapidly multiplying
stock plant material to produce a large number of progeny
plants, using modern plant tissue culture methods.

3. Micropropagation is used to multiply noble plants such as
those that have been genetically modified or bred through
conventional plant breeding methods. It is also used to
provide a sufficient number of plantlets for planting from a
stock plant which does not produce seeds, or does not
respond well to vegetative reproduction.
Technique of Micropropagation:
 Micro propagation is a complicated process and mainly
involves 5 stages (stage 0 and IV) for more comprehensive
representation of micro- propagation.
 Stage 0:This is the initial step in micropropagation, and
involves the selection and growth of stock plants for about
3 months under controlled conditions.
 Stage I: In this stage, the initiation and establishment of
culture in a suitable medium is achieved. Selection of
appropriate explants is important.The most commonly used
explants are organs, shoot tips and axillary buds.

4.  Stage II: In this stage, the major activity of micro
propagation occurs in a defined culture medium.
Stage II mainly involves multiplication of shoots or
rapid embryo formation from the explant.
 Stage III:This stage involves the transfer of shoots to
a medium for rapid development into shoots.
Sometimes, the shoots are directly planted in soil to
develop roots. In vitro rooting of shoots is preferred
while simultaneously handling a large number of
species.
 Stage IV:This stage involves the establishment of
plantlets in soil.This is done by transferring the
plantlets of stage III from the laboratory to the
environment of greenhouse. For some plant species,
stage III is skipped, and un-rooted stage II shoots are
planted in pots or in suitable compost mixture.

5. StageV: In this stage, the major activity of micro
propagation occurs in a defined culture medium.
Stage II mainly involves multiplication of shoots or
rapid embryo formation from the explant.

6. Stages involved in micropropagation

7. Approaches involved in
Micropropagation
1. Multiplication by axillary buds/apical shoots.
2. Multiplication by adventitious shoots. Besides
the above two approaches, the plant regeneration
processes namely organogenesis and somatic
embryo- genesis may also be treated as micro
propagation.
3. Organogenesis:The formation of individual organs
such as shoots, roots, directly from an explant or
from the callus and cell culture induced from the
explant.
4. Somatic embryogenesis:The regeneration of
embryos from somatic cells, tissues or organs.

8. 1 Multiplication by axillary buds/apical shoots.
Actively dividing meristems are present at the axillary buds and
apical shoots (shoot tips).
=>The axillary buds located in the axils of leaves are capable of
developing into shoots.
=>By means of induced in vitro multiplication in micro
propagation, it is possible to develop plants from meristem and
shoot tip cultures and from bud cultures.

9. Shoot tip cultures-
Apical meristem is a dome of tissue located at the
extreme tip of a shoot.The apical meristem along with
the young leaf primordia constitutes the shoot apex.

10. Meristem or shoot tip is isolated from a stem by a V-
shaped cut.The size (frequently 0.2 to 0.5 mm) of the
tip is critical for culture. In general, the larger the
explant (shoot tip), the better are the chances for
culture survival. For good results of micro propagation,
explants should be taken from the actively growing
shoot tips, and the ideal timing is at the end of the
plants dormancy period.
 The most widely used media for meristem culture are
MS medium andWhite’s medium.

12.  In stage I, the culture of meristem is established.
Addition of growth regulators namely cytokinins
(kinetin, BA) and auxins (NAA or IBA) will support the
growth and development.
In stage II, shoot development along with axillary shoot
proliferation occurs. High levels of cytokinins are
required for this purpose.
Stage III is associated with rooting of shoots and further
growth of plantlet.
Consequently, stage II medium and stage III medium
should be different in composition.The optimal
temperature for culture is in the range of 20-28°C (for
majority 24-26°C). Lower light intensity is more
appropriate for good micro propagation.

13. Bud Cultures-
The plant buds possess quiescent or active
meristems depending on the physiological state of the
plant.Two types of bud cultures are used— single node
culture and axillary bud culture.
Single node culture : This is a natural method for
vegetative propagation of plants both in vivo and in
vitro conditions. A bud along with a piece of stem is
isolated and cultured to develop into a plantlet. Closed
buds are used to reduce the chances of infections. In
single node culture, no cytokinin is added.

16. Axillary bud culture: In this method, a shoot tip
along with axillary bud is isolated.The cultures are
carried out with high cytokinin concentration. As a
result of this, apical dominance stops and axillary buds
develop. A schematic representation of axillary bud
culture for a rosette plant and an elongate
plantisgivenas follows.

17. For a good axillary bud culture, the cytokinin/ auxin
ratio is around 10: 1.
This is however, variable and depends on the nature
of the plant species and the developmental stage
of the explant used. In general, juvenile explants
require less cytokinin compared to adult explants.
Sometimes, the presence of apical meristem may
interfere with axillary shoot development. In such
a case, it has to be removed.

18. 2 Multiplication by adventitious
shoots
 The stem and leaf structures that are naturally
formed on plant tissues located in sites other
than the normal leaf axil regions are regarded as
adventitious shoots.
 There are many adventitious shoots which
include stems, bulbs, tubers and rhizomes.The
adventitious shoots are useful for in vivo and in
vitro clonal propagation.
 The meristematic regions of adventitious shoots
can be induced in a suitable medium to
regenerate to plants.

20. 3 Organogenesis
 Organogenesis is the process of morphogenesis involving the
formation of plant organs i.e. shoots, roots, flowers, buds from
explant or cultured plant tissues. It is of two types i.e direct
organogenesis and indirect organogenesis.
 Direct organogenesis-Tissues from leaves, stems, roots and
inflorescences can be directly cultured to produce plant organs.
In direct organogenesis, the tissue undergoes morphogenesis
without going through a callus or suspension cell culture stage.
 Indirect organogenesis-When the organogenesis occurs
through callus or suspension cell culture formation, it is
regarded as indirect organogenesis. Callus growth can be
established from many explants (leaves, roots, cotyledons,
stems, flower petals etc.) for subsequent organogenesis.

23. In general, the bigger the explant the better the
chances for obtaining viable callus/cell
suspension cultures. It is advantageous to select
meristematic tissues (shoot tip, leaf, and petiole)
for efficient indirect organogenesis.This is
because their growth rate and survival rate are
much better.
For indirect organogenesis, the cultures may be
grown in liquid medium or solid medium. Many
culture media (MS, B5 White’s etc.) can be used
in organogenesis.The concentration of growth
regulators in the medium is critical for organo-
genesis.

24. 4. Embryogenesis
The process of regeneration of embryos from somatic
cells, tissues or organs is regarded as somatic (or
asexual) embryogenesis.
Somatic embryos are structurally similar to zygotic
(sexually formed) embryos, and they can be excised
from the parent tissues and induced to germinate in
tissue culture media.
Two routes of somatic embryogenesis are known —
direct and indirect.
 Direct Somatic Embryogenesis-When the somatic
embryos develop directly on the excised plant (explant)
without undergoing callus formation, it is referred to as
direct somatic embryogenesis .This is possible due to the
presence of pre-embryonic determined cells (PEDQ found
in certain tissues of plants.

25. • Indirect Somatic Embryogenesis- In
indirect embryogenesis, the cells from explant
(excised plant tissues) are made to proliferate
and form callus, from which cell suspension
cultures can be raised. Certain cells referred to
as induced embryo genic determined cells
(IEDC) from the cell suspension can form
somatic embryos.
Embryogenesis is made possible by the
presence of growth regulators (in appropriate
concentration) and under suitable
environmental conditions.

27.  Somatic embryogenesis (direct or indirect) can be
carried on a wide range of media (e.g.
MS,White’s).The addition of the amino acid L-
glutamine promotes embryogenesis.The presence of
auxin such as 2, 4- dichlorophenoxy acetic acid is
essential for embryo initiation. On a low auxin or no
auxin medium, the embryo genic clumps develop
into mature embryos.
 Indirect somatic embryogenesis is commercially
very attractive since a large number of embryos can
be generated in a small volume of culture
medium.The somatic embryos so formed are
synchronous and with good regeneration capability.

28. Applications of Micro propagation
Micro propagation has become a suitable alternative
to conventional methods of vegetative propagation
of plants.There are several advantages of micro
propagation.
 High Rate of Plant Propagation- Through micro
propagation, a large number of plants can be
grown from a piece of plant tissue within a short
period. Another advantage is that micro
propagation can be carried out throughout the
year, irrespective of the seasonal variations.
 Production of Disease-free Plants- It is possible
to produce disease-free plants through micro
propagation. Meristem tip cultures are generally
employed to develop pathogen-free plants .

29.  Cost-effective Process- Micro propagation requires
minimum growing space.Thus, millions of plant
species can be maintained inside culture vials in a
small room in a nursery.
 Production of seeds in some crops- Micro
propagation, through axillary bud prolife- ration
method, is suitable for seed production in some
plants. This is required in certain plants where the
limitation for seed production is high degree of
genetic conservation e.g. cauliflower, onion.
 It is the only viable method of regenerating
genetically modified cells after protoplast fusion.
Micropropagation often produces more robust
plants, leading to accelerated growth compared to
similar plants produced by conventional methods -
like seeds or cuttings.

30. Disadvantages of Micropropagation
 Contamination of Cultures- During the course of
micro propagation, several slow-growing microorganisms
(e.g. Eswinia sp, Bacillus sp) contaminate and grow in
cultures. The microbial infection can be controlled by
addition of antibiotics or fungicides.
 Brewing of Medium- Micro propagation of certain
plants (e.g. woody perennials) is often associated with
accumulation of growth inhibitory substances in the
medium. Chemically, these substances are phenolic
compounds, which can turn the medium into dark colour.
Phenolic compounds are toxic and can inhibit the growth
of tissues. Brewing of the medium can be prevented by
the addition of ascorbic acid or citric acid or polyvinyl
pyrrolidone to the medium.

31. Disadvantages of Micropropagation
GeneticVariability -When micro propagation is carried out
through shoot tip cultures, genetic variability is very low.
However, use of adventitious shoots is often associated
with pronounced genetic variability.
 Vitrification-During the course of repeated in vitro shoot
multiplication, the cultures exhibit water soaked or almost
translucent leaves. Such shoots cannot grow and even may
die.This phenomenon is referred to as vitrification. This can
be be prevented by increasing the agar concentration (from
0.6 to 1%) in the medium. However, increased agar
concentration reduces the growth rate of tissues.
 For some micro propagation techniques, expensive
equipment, sophisticated facilities and trained manpower
are needed.This limits its use.